Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash- absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840+, together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840+ was reduced by cytochrome c with a t(1/2) of ≃65 μs after the first flash, 80% with a t(1/2) of ≃100 μs after the second flash, and 23% with a t(1/2) of ≃100 μs after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change ΔG°of -53 meV for electron transfer from one cytochrome c to P840+, ΔG°being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes C which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 μs. No recombination reaction between the primary partners P840+ and A0 - could be detected under normal conditions. Such a recombination was observed (t(1/2) ≃19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.
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